Malignant melanoma is notoriously resistant to cytotoxic therapies. However, both spontaneous remissions and durable responses to a variety of immunotherapy strategies occur in melanoma. The adoptive transfer of cloned antigen-specific T cells, a laborious technique, has resulted in the highest response rates. In order to make this mode of melanoma immunotherapy more widely applicable, a strategy that minimizes ex vivo manipulation of cells would be desirable. Additionally, clinical development of such a strategy relies on the availability of assays to efficiently study the performance of tumor antigen-specific cells in vivo. The monitoring of immune responses to cancer is currently based on ex vivo assays; e.g. MHC tetramer and ELISPOT assays, which use lymphocytes sampled from peripheral blood. Their use as surrogate endpoints for the clinical development of immunotherapy strategies has obvious caveats, the main ones being (i) the sampling of cells at the wrong compartment (the blood and not the tumor) and (ii) not allowing a dynamic evaluation of the T cell responses. We hypothesized that lymphocytes engineered to express both a transgenic T cell receptor (TCR) specific for a defined melanoma antigen and a PET reporter gene can be used to study non-invasively the in vivo kinetics of antitumor T cell responses. This system would allow the efficient generation of a population of antigen-specific T cells labeled with a PET reporter gene for adoptive transfer, which could then be tracked in vivo by serial PET scanning to determine their population dynamics and ability to home to antigen-matched tumors. We propose a 3-aim project that builds upon the experience in the in vivo imaging of antigen-specific T cell responses in mice generated during the current UCLA ICMIC funding period. In Aim 1 we plan to construct and test in vitro retroviral and lentiviral vectors expressing the TCR for the melanoma antigen Tyrosinase (Tyr) and the HSV1-sr39tk PET reporter gene. In Aim 2 we propose a murine model to image the in vivo kinetics of genetically modified murine splenocytes and hematopoietic stem cells (HSC) expressing both the Tyr-TCR and HSV1-sr39tk. We will serially image their ability to repopulate lymphopenic hosts upon adoptive transfer, and traffic to antigen-matched experimental tumors in vivo. In Aim 3 we plan a phase I clinical trial in patients with metastatic melanoma. In this trial, we propose to administer increasing doses of Tyr-TCR-HSV1-sr39tk transgenic autologous cells after a non-myeloablative conditioning regimen and determine, by PET imaging, their ability to accumulate in Tyr-positive melanoma metastasis. In summary, we propose the preclinical and clinical testing of the ability to image tumor antigen-specific T cell responses against malignant melanoma.